Placental tissue grafts

ABSTRACT

Described herein are tissue grafts derived from the placenta. The grafts are composed of at least one layer of amnion tissue where the epithelium layer has been substantially removed in order to expose the basement layer to host cells. By removing the epithelium layer, cells from the host can more readily interact with the cell-adhesion bio-active factors located onto top and within of the basement membrane. Also described herein are methods for making and using the tissue grafts. The laminin structure of amnion tissue is nearly identical to that of native human tissue such as, for example, oral mucosa tissue. This includes high level of laminin-5, a cell adhesion bio-active factor show to bind gingival epithelia-cells, found throughout upper portions of the basement membrane.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuing application of U.S. application Ser.No. 12/428,908, filed on Apr. 23, 2009, which is a continuationapplication of U.S. application Ser. No. 12/206,508, filed on Sep. 8,2008, which claims priority upon U.S. provisional application Ser. Nos.60/970,780, filed Sep. 7, 2007; 60/986,665, filed Nov. 9, 2007; and60/989,299, filed Nov. 20, 2007. Each application is hereby incorporatedby reference in its entirety for all of its teachings.

BACKGROUND

Human placental membrane (e.g. amniotic membrane or tissue) has beenused for various types of reconstructive surgical procedures since theearly 1900s. The membrane serves as a substrate material, more commonlyreferred to as a biological dressing or patch graft. Such a membrane hasalso been used widely for ophthalmic procedures. Typically, suchmembrane is either frozen or dried for preservation and storage untilneeded for surgery.

Such placental tissue is typically harvested after an elective Cesareansurgery. The placenta is composed of the umbilical cord and amnioticsac. The amniotic sac, commonly referred to as the amniotic membrane,has two primary layers of tissue, amnion and chorion. Amnion tissue isinnermost layer of the amniotic sac and in direct contact with theamniotic fluid. The amniotic sac contains the amniotic fluid andprotects the fetal environment. Histological evaluation indicates thatthe membrane layers of the amnion consist of single layer of epitheliumcells, thin reticular fibers (basement membrane), a thick compact layer,and fibroblast layer. The fibrous layer of amnion (i.e., the basementmembrane) contains collagen types IV, V, and VII, and cell-adhesionbio-active factors including fibronectin and laminins

Amnion tissue provides unique grafting characteristics when used forsurgical procedures, including providing a matrix for cellularmigration/proliferation, providing a natural biological barrier, arenon-immunogenic, and contains numerous bio-active molecules, which canbe used as a membrane to assist in tissue regeneration and improvedhealing outcomes in numerous applications. The membrane has thecapability to self-adhere or, in the alternative, is susceptible ofbeing fixed in place using different techniques including fibrin glue orsuturing. Such grafts, when properly prepared, can be stored at roomtemperature for extended periods of time, without need for refrigerationor freezing, until needed for a surgical procedure.

Known clinical procedures or applications for such amnion grafts includeocular reconstruction, burns, anti-adhesion applications, barriermembranes, and general wound care. Described herein are membranesutilizing amnion tissue as building blocks to build membranes wellsuited for use in a variety of application such as, for example,perioplastic surgery and other surgical applications involving humanmucosa tissue. The grafts and methods described herein utilize thesebiological molecules to enhance the performance of the amnioticmembrane.

SUMMARY OF INVENTION

Described herein are tissue grafts derived from the placenta. The graftsare composed of at least one layer of amnion tissue where the epitheliumlayer has been substantially removed in order to expose the basementlayer to host cells. By removing the epithelium layer, cells from thehost can more readily interact with the cell-adhesion bio-active factorslocated onto top and within of the basement membrane. Also describedherein are methods for making and using the tissue grafts. The lamininstructure of amnion tissue is nearly identical to that of native humantissue such as, for example, oral mucosa tissue. This includes highlevel of laminin-5, a cell adhesion bio-active factor show to bindgingival epithelia-cells, found throughout upper portions of thebasement membrane. The advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the aspects describedbelow. The advantages described below will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1 is an overview flow chart of the process for making the tissuegrafts described herein.

FIG. 2 is an exemplary tissue check-in form used with the tissue graftsdescribed herein.

FIG. 3 is an exemplary raw tissue assessment form used with the tissuegrafts described herein.

FIG. 4 is an exemplary dehydration process form used with the tissuegrafts described herein.

FIG. 5 is a perspective view of an exemplary drying fixture for makingthe tissue grafts described herein.

FIGS. 6-9 are representative side views of different tissue graftsdescribed herein.

FIG. 10 is a top view of a tissue graft with perforations.

DETAILED DESCRIPTION

Before the present articles and methods are disclosed and described, itis to be understood that the aspects described below are not limited tospecific compounds, synthetic methods, or uses as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular aspects only and is notintended to be limiting.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a bioactive agent” includes mixtures of two or more suchagents, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally cleaning step” means thatthe cleaning step may or may not be performed.

Titles or subtitles may be used in the specification for the convenienceof a reader, which are not intended to influence the scope of thepresent invention. Additionally, some terms used in this specificationare more specifically defined below.

I. Tissue Grafts and Methods for Making Thereof

Described herein are tissue grafts and methods of making and usingthereof. In general, the tissue grafts are multilayered systems composedof one or more membranes laminated to a base amnion, where theepithelium layer of the base amnion has been substantially removed. Inone aspect, the process for preparing the amnion and removing theepithelium layer involves

-   (a) obtaining a placenta from a subject, wherein the placenta    comprises an amniotic membrane layer and a chorion tissue layer;-   (b) separating the chorion tissue layer from the amnion layer,    wherein the amnion comprises epithelium cells adjacent to a basement    membrane;-   (d) removing substantially all of the epithelium cells to expose the    basement membrane of the amnion;-   (e) mounting the first membrane onto a surface of a drying fixture;-   (f) mounting one or more additional membranes on the first membrane    to produce a layered tissue graft; and-   (g) dehydrating the layered tissue graft on the drying fixture.    FIG. 1 depicts an overview (100) of the steps to harvest, process,    and prepare placental material for later use as a tissue graft is    disclosed. More detailed descriptions and discussion regarding each    individual step will follow. Initially, the placenta tissue is    collected from a consenting patient following an elective Cesarean    surgery (step 110). The material is preserved and transported in    conventional tissue preservation manner to a suitable processing    location or facility for check-in and evaluation (step 120). Gross    processing, handling, and separation of the tissue layers then takes    place (step 130). After the epithelium layer is substantially    removed from the amnion to expose the base membrane (step 135),    acceptable tissue is then decontaminated (step 140), dehydrated    (step 150), cut and packaged (step 160), tissue is radiological    terminal sterilized using gamma radiation (step 165), and released    (step 170) to the market for use by surgeons and other medical    professionals in appropriate surgical procedures and for wound care.    Each step is described in detail below.    Initial Tissue Collection (Step 110)

The recovery of placenta tissue originates in a hospital, where it iscollected during a Cesarean section birth. The donor, referring to themother who is about to give birth, voluntarily submits to acomprehensive screening process designed to provide the safest tissuepossible for transplantation. The screening process preferably tests forantibodies to the human immunodeficiency virus type 1 and type 2(anti-HIV-1 and anti-HIV-2), hepatitis B surface antigens (HBsAg),antibodies to the hepatitis C virus (anti-HCV), antibodies to the humanT-lymphotropic virus type I and type II (anti-HTLV-I and anti-HTLV-II),CMV, and syphilis, using conventional serological tests. The above listof tests is exemplary only, as more, fewer, or different tests may bedesired or necessary over time or based upon the intended use of thegrafts, as will be appreciated by those skilled in the art.

Based upon a review of the donor's information and screening testresults, the donor will either be deemed acceptable or not. In addition,at the time of delivery, cultures are taken to determine the presence ofbacteria, for example, Clostridium or Streptococcus. If the donor'sinformation, screening tests, and the delivery cultures are allsatisfactory (i.e., do not indicate any risks or indicate acceptablelevel of risk), the donor is approved by a medical director and thetissue specimen is designated as initially eligible for furtherprocessing and evaluation.

Human placentas that meet the above selection criteria are preferablybagged in a saline solution in a sterile shipment bag and stored in acontainer of wet ice for shipment to a processing location or laboratoryfor further processing.

If the placenta tissue is collected prior to the completion or obtainingof results from the screening tests and delivery cultures, such tissueis labeled and kept in quarantine. The tissue is approved for furtherprocessing only after the required screening assessments and deliverycultures, which declare the tissue safe for handling and use, aresatisfied and obtains final approval from a medical director.

Material Check-in and Evaluation (Step 120)

Upon arrival at the processing center or laboratory, the shipment isopened and verified that the sterile shipment bag/container is stillsealed and in the coolant, that the appropriate donor paperwork ispresent, and that the donor number on the paperwork matches the numberon the sterile shipment bag containing the tissue. The sterile shipmentbag containing the tissue is then stored in a refrigerator until readyfor further processing. All appropriate forms, including a tissuecheck-in form, such as that shown in FIG. 2, are completed and chain ofcustody and handling logs (not shown) are also completed.

Gross Tissue Processing (Step 130)

When the tissue is ready to be processed further, the sterile suppliesnecessary for processing the placenta tissue further are assembled in astaging area in a controlled environment and are prepared forintroduction into a controlled environment. If the controlledenvironment is a manufacturing hood, the sterile supplies are opened andplaced into the hood using conventional sterile technique. If thecontrolled environment is a clean room, the sterile supplies are openedand placed on a cart covered by a sterile drape. All the work surfacesare covered by a piece of sterile drape using conventional steriletechniques, and the sterile supplies and the processing equipments areplaced on to the sterile drape, again using conventional steriletechniques.

Processing equipment is decontaminated according to conventional andindustry-approved decontamination procedures and then introduced intothe controlled environment. The equipment is strategically placed withinthe controlled environment to minimize the chance for the equipment tocome in proximity to or is inadvertently contaminated by the tissuespecimen.

Next, the placenta is removed from the sterile shipment bag andtransferred aseptically to a sterile processing basin within thecontrolled environment. The sterile basin contains hyperisotonic salinesolution (e.g., 18% NaCl) that is at room or near room temperature. Theplacenta is gently massaged to help separate blood clots and to allowthe placenta tissue to reach room temperature, which will make theseparation of the amnion and chorion layers from each other, asdiscussed hereinafter, easier. After having warmed up to the ambienttemperature (after about 10-30 minutes), the placenta is then removedfrom the sterile processing basin and laid flat on a processing traywith the amniotic membrane layer facing down for inspection.

The placenta tissue is examined and the results of the examination aredocumented on a “Raw Tissue Assessment Form” similar to that shown inFIG. 3. The placenta tissue is examined for discoloration, debris orother contamination, odor, and signs of damage. The size of the tissueis also noted. A determination is made, at this point, as to whether thetissue is acceptable for further processing.

Next, if the placenta tissue is deemed acceptable for furtherprocessing, the amnion and chorion layers of the placenta tissue arethen carefully separated. In one aspect, the materials and equipmentused in this procedure include a processing tray, 18% saline solution,sterile 4×4 sponges, and two sterile Nalgene jars. The placenta tissueis then closely examined to find an area (typically a corner) in whichthe amnion layer can be separated from the chorion layer. The amnionappears as a thin, opaque layer on the chorion.

Prior to removal of the epithelium, the fibroblast layer is identifiedby gently contacting each side of the membrane with a piece of sterilegauze or a cotton tipped applicator. The fibroblast layer will stick tothe test material. The amnion is placed into processing tray fibroblastlayer down. Using a blunt instrument, a cell scraper or sterile gauze,any residual blood is also removed. This step must be done with adequatecare, again, so as not to tear the amnion or chorion tissues. Thecleaning of the amnion is complete once the amnion tissue is smooth andopaque-white in appearance. If the amnion tissue is cleaned too much,the jelly-like fibroblast layer can be removed. Any areas of the amnioncleaned too aggressively and appear clear will be unacceptable and willultimately be discarded.

Removal of Epithelium Layer from Amnion (Step 135)

The epithelium layer present on the amnion is substantially removed inorder to expose the basement layer of the amnion. The significance ofremoving the epithelium layer is described below. The term“substantially removed” with respect to the amount of epithelium removedis defined herein as removing greater than 90%, greater than 95%, orgreater than 99% of the epithelial cells from the amnion. The presenceor absence of epithelial cells remaining on the amnion layer can beevaluated using techniques known in the art. For example, after removalof the epithelial cell layer, a representative tissue sample from theprocessing lot is placed onto a standard microscope examination slide.The tissue sample is then stained using Eosin Y Stain and evaluated asdescribed below. The sample is then covered and allowed to stand. Oncean adequate amount of time has passed to allow for staining, visualobservation is done under magnification.

The epithelium layer can be removed by techniques known in the art. Forexample, the epithelium layer can be scraped off of the amnion using acell scraper. Other techniques include, but are not limited to, freezingthe membrane, physical removal using a cell scraper, or exposing theepithelial cells to nonionic detergents, anionic detergents, andnucleases. The de-epithelialized tissue is then evaluated to determinethat the basement membrane has not been compromised and remains intact.This step is performed after completion of the processing step and thetissue has been dehydrated as described in the next section. Forexample, a representative sample graft is removed for microscopicanalysis. The tissue sample is place onto a standard slide and 100 μl ofEosin Y stain is applied to the sample and allowed to set. The tissuesample is then examined under magnification. Cellular material willstain dark indicating the presence of cells. If no stained cells arepresent, de-epithelization has been achieved.

Chemical Decontamination (Step 140)

The amnion is then placed into a sterile Nalgene jar for the next stepfor additional cleaning If the chorion is to be recovered and processedfurther, it too is placed in its own sterile Nalgene jar for additionalcleaning If the chorion is not to be kept or used further, it can bediscarded in an appropriate biohazard container. In one aspect, thefollowing procedure can be used to clean the amnion.

Each Nalgene jar is aseptically filled with 18% saline hypertonicsolution and sealed (or sealed with a top). The jar is then placed on arocker platform and agitated for between 30 and 90 minutes, whichfurther cleans the tissue of contaminants. If the rocker platform wasnot in the critical environment (e.g., the manufacturing hood), theNalgene jar is returned to the controlled/sterile environment andopened. Using sterile forceps or by aseptically decanting the contents,the tissue is gently removed from the Nalgene jar containing the 18%hyperisotonic saline solution and placed into an empty Nalgene jar. Thisempty Nalgene jar with the tissue is then aseptically filled with apre-mixed antibiotic solution. Preferably, the premixed antibioticsolution is comprised of a cocktail of antibiotics, such as StreptomycinSulfate and Gentamicin Sulfate. Other antibiotics, such as Polymixin BSulfate and Bacitracin, or similar antibiotics now available oravailable in the future, are also suitable. Additionally, it ispreferred that the antibiotic solution be at room temperature when addedso that it does not change the temperature of or otherwise damage thetissue. This jar or container containing the tissue and antibiotics isthen sealed or closed and placed on a rocker platform and agitated for,preferably, between 60 and 90 minutes. Such rocking or agitation of thetissue within the antibiotic solution further cleans the tissue ofcontaminants and bacteria.

Again, if the rocker platform was not in the critical environment (e.g.,the manufacturing hood), the jar or container containing the tissue andantibiotics is then returned to the critical/sterile environment andopened. Using sterile forceps, the tissue is gently removed from the jaror container and placed in a sterile basin containing sterile water ornormal saline (0.9% saline solution). The tissue is allowed to soak inplace in the sterile water/normal saline solution for at least 10 to 15minutes. The tissue may be slightly agitated to facilitate removal ofthe antibiotic solution and any other contaminants from the tissue.After at least 10 to 15 minutes, the tissue is ready to be dehydratedand processed further.

In the case when the chorion is to be used, the following exemplaryprocedure can be used. After separation of the chorion from the amnionand removal of clotted blood from the fibrous layer, the chorion isrinsed in 18% saline solution for 30 min. During the first rinse cycle,18% saline is heated in a sterile container using laboratory heatingplate such that the solution temperature is approximately 48° C. Thesolution is decanted, the chorion tissue is placed into the sterilecontainer, and decanted saline solution is poured into the container.The container is sealed and placed on rocker plate and agitated for 1hour. After 1 hour agitation bath, remove the tissue and place thetissue into second heated agitation bath for an additional 1 hour rinsecycle. Next, the chorion tissue is placed into 200 ml of 0.5% Triton-Xwash solution. The container is sealed and agitated without heat for 2hours. The tissue is next washed with deionized water (250 ml of DIwater×4) with vigorous motion for each rinse. The tissue is removed andplaced into a container of 1× PBS w/EDTA solution. The container issealed and agitated for 1 hour at controlled temperature for 8 hours.The tissue is removed and rinsed using sterile water. A visualinspection was performed to remove any remaining discolored fibrousblood material from the membrane. Membrane should have a cream whitevisual appearance with no evidence of brownish discoloration.

Preparation of Tissue Graft and Dehydration (Step 150)

After removal of the epithelium layer, the amnion is ready to be used toproduce the tissue graft. In one aspect, the amnion layer is laid on asuitable drying fixture, where the exposed basement membrane is adjacentto the surface of the drying fixture. In other aspects, the amnion canbe placed on the surface of the drying fixture such that the exposedbasement membrane is facing up. The drying fixture is preferably sizedto be large enough to receive the amnion tissue, fully, in laid out,flat fashion. In one aspect, the drying fixture is made of Teflon or ofDelrin, which is the brand name for an acetal resin engineering plasticinvented and sold by DuPont and which is also available commerciallyfrom Werner Machine, Inc. in Marietta, Ga. Any other suitable materialthat is heat and cut resistant, capable of being formed into anappropriate shape to receive wet tissue and to hold and maintaintextured designs, logos, or text can also be used for the dryingfixture. It is desirable that the amnion tissue be placed on the dryingfixture so that it completely covers as many “product spaces” (asexplained hereinafter) as possible.

In one aspect, similar to that shown in FIG. 5, the receiving surface ofthe drying fixture 500 has grooves 505 that define the product spaces510, which are the desired outer contours of the tissue after it is cutand of a size and shape that is desired for the applicable surgicalprocedure in which the tissue will be used. For example, the dryingfixture can be laid out so that the grooves are in a grid arrangement.The grids on a single drying fixture may be the same uniform size or mayinclude multiple sizes that are designed for different surgicalapplications. Nevertheless, any size and shape arrangement can be usedfor the drying fixture, as will be appreciated by those skilled in theart. In another embodiment, instead of having grooves to define theproduct spaces, the drying fixture has raised ridges or blades.

Within the “empty” space between the grooves or ridges, the dryingfixture can include a slightly raised or indented texture in the form oftext, logo, name, or similar design 520. This textured text, logo, name,or design can be customized. When dried, the tissue will mold itselfaround the raised texture or into the indented texture—essentiallyproviding a label within the tissue itself. Preferably, suchtexture/label can be read or viewed on the tissue in only oneorientation so that, after drying and cutting, an end user (typically, aclinician) of the dried tissue will be able to tell the stromal sidefrom the basement side of the dried tissue. The reason this is desiredis because, during a surgical procedure, it is desirable to place theallograft in place, with basement side down or adjacent the nativetissue of the patient receiving the allograft. FIG. 5 illustrates avariety of marks, logos, and text 520 that can be included within theempty spaces 510 of the drying fixture 500. Typically, a single dryingfixture will include the same design or text within all of the emptyspaces; however, FIG. 5 shows, for illustrative purposes, a wide varietyof designs that can be included on such drying fixtures to emboss eachgraft. After the layer of amnion with substantially no epithelial cellson the surface of the basement membrane has been applied to the dryingfixture, one or more membranes can applied to the base amnion layer toproduce the tissue graft. FIGS. 6-9 depict numerous examples ofmulti-laminated tissue grafts produced by the methods described herein.Referring to FIG. 6, amnion layer 610 is applied to the surface of thedrying fixture 600, where the basement membrane 605 is adjacent to thesurface of the drying fixture 600. Two additional amnion layers 620 and630 are applied to amnion 610. In this aspect, the fibrous layer of eachamnion layer acts as an adhesive for the next layer. Thus, the fibrouslayer of amnion 610 adheres to the epithelial layer of amnion 620. It isimportant to note that when multiple amnion layers are used, it is notnecessary to remove the epithelial cells from the basement membrane forthose layers that are not in direct contact with host cells. Although inthis aspect, the fibroblast layer is used to adhere the membranestogether, other techniques and materials such as, for example, fibringlue, gelatin, photochemical techniques, and suturing can be used toproduce the multi-laminated tissue graft.

FIGS. 7 and 8 depict additional features of the multi-layered tissuegraft. Referring to FIG. 7, amnion layer 700 minus the epithelial layeris applied to drying fixture 600. Four additional amnion layers(710-740) are then applied to the amnion layer 700. In FIG. 8, amnionlayer 800 minus the epithelial layer is applied to drying fixture 600,and chorion layer 810 is applied to the amnion layer 800.

In another aspect, it is also contemplated that the multi-layered tissuegraft can be composed of one or more membranes sandwiched between twoamnion membranes with the amnion tissue with its epithelial layerremoved exposing the basement membrane of the amnion tissue to hostcells. This aspect is depicted in FIG. 9, where amnion layers 920 and930 are sandwiched between amnion layers 910 and 940 (no epitheliallayer).

Although FIGS. 6-9 depict the use of amnion and chorion to produce themulti-layered tissue graft, other biodegradable biologically compatiblematerials can be used in place of chorion to form a multi-layered amnioncomposite membrane. Examples of such materials include, but are notlimited to, allograft pericardium, allograft acellular dermis, amnioticmembrane (i.e., both amnion and chorion), Wharton's jelly, purifiedxenograft Type-1 collagen, biocellulose polymers or copolymers,biocompatible synthetic polymer or copolymer films, purified smallintestinal submucosa, bladder acellular matrix, cadaveric fascia, or anycombination thereof.

The actual number of layers will depend upon the surgical need andprocedure with which the tissue graft is designed to be used for. Forexample, in periodontal applications such as root coverage procedures, asingle membrane that is between 20-50 μm in thickness to multilayertissue grafts having a thickness up to 2 mm can be used. In one aspect,the number of membranes laminated to the base amnion tissue withsubstantially all of the epithelial cells removed can be one, two, fiveand or ten, with the ultimate number of layers dependent of the type ofmembrane used, and the expected indication for use.

The multi-layered tissue grafts described herein are thicker andstronger than a single layer of base amnion. The base amnion may havehandling limitations. In general, tissue grafts are cut to size to matchthe morphology of the wound, placed on or within the wound, and ifdesired, can be held in place with sutures. If left unsupported, thebase amnion tissue may fall over itself, and may bunch together.Moreover, the native amnion tissue may be unable to hold sutures.Sutures may tear the tissue during placement, and may be knocked free bythe patient. These issues make trimming, placement, and securing asingle layer of base amnion tissue less than ideal for the medicalprofessional.

Once the tissue graft is placed on the drying fixture, in one aspect,the drying fixture is placed in a sterile Tyvex (or similar, breathable,heat-resistant, and sealable material) dehydration bag and sealed. Sucha breathable dehydration bag prevents the tissue from drying too quicklyand prevents cross-contamination, when multiple donors are dehydrated inthe oven at the same time. If multiple drying fixtures are beingprocessed simultaneously, each drying fixture is either placed in itsown Tyvex bag or, alternatively, placed into a suitable mounting framethat is designed to hold multiple drying frames thereon and the entireframe is then placed into a larger, single sterile Tyvex dehydration bagand sealed.

The Tyvex dehydration bag containing the one or more drying fixtures isthen placed into an oven or incubator (vacuum or non-vacuum) that hasbeen preheated to approximately 35 to 50° C. The Tyvex bag remains inthe oven for between 30 and 120 minutes, although approximately 45minutes at a temperature of approximately 45° C. appears to be ideal todry the tissue sufficiently but without over-drying the tissue. Thespecific temperature and time for any specific oven will need to becalibrated and adjusted based on other factors including altitude, sizeof the oven, accuracy of the oven temperature, material used for thedrying fixture, number of drying fixtures being dried simultaneously,whether a single or multiple frames of drying fixtures are driedsimultaneously, and the like. In one aspect, the dehydration recordationform, similar to that shown in FIG. 4, can be completed at the end ofthe dehydration process.

In certain aspects the membrane is not physically altered except forfinal cutting and packaging (step 160). When completed, the processedtissue graft has a semi-transparent appearance with a whitishcoloration. The tissue is pliable to withstand bending and sizing in itsdry, non-hydrated state. The tissue grafts described herein can bestored at room temperature for extended periods of time.

In certain aspects, after the dehydration phase, the tissue graft isperforated using a small gauge needle, or punch to produce a mesh-typeconfiguration, as shown in FIG. 10, where a plurality of holes 1010 arepresent in graft 1000. The size and spacing of the holes can varydepending upon the application. In one aspect, the holes have a diameterfrom (0.2 mm-2.0 mm or 0.2 mm to 0.5 mm) and are spaced from 1 mm to 5mm or 2 mm to 4 mm apart. The holes may facilitate cell migration andprovide for the passage of blood and other factors to the opposite sideof the graft, which ultimately enhances wound healing. For example, inseveral perioplastic procedures, the membrane is placed directly ontothe exposed root of the tooth. Here, the tooth surface does not supply asource of blood and the holes provide a source of blood and otherfactors to the side of the membrane that is in direct contact with thetooth surface. In multi-layered products, the holes may allow for a morerapid and even distribution of wound healing agents which may be appliedand or adsorbed onto prior to implantation to further improve theregenerative potential of the treatment. When completed, the processedtissue graft has a semi-transparent appearance with a whitishcoloration. The tissue is pliable to withstand bending and sizing in itsdry, non-hydrated state. The tissue grafts described herein can bestored at room temperature for extended periods of time.

Cutting & Packaging (Step 160)

Once the tissue graft has been adequately dehydrated, the tissue graftis then ready to be cut into specific product sizes and appropriatelypackages for storage, terminal sterilization, and later surgical use. Inone aspect, the Tyvex bag containing the dehydrated tissue is placedback into the sterile/controlled environment. The number of grafts to beproduced is estimated based on the size and shape of the tissue on thedrying fixture(s). An appropriate number of pouches, one for each tissuegraft, are then also introduced into the sterile/controlled environment.The drying fixture(s) are then removed from the Tyvex bag.

If the drying fixture has grooves, then the following exemplaryprocedure is followed for cutting the tissue into product sizes. If thedrying fixture is configured in a grid pattern, a #20 or similarstraight or rolling blade is used to cut along each groove line inparallel. Then, all lines in the perpendicular direction are cut.Alternatively, if the drying fixture has raised edges or blades, thenthe following procedure is followed for cutting the tissue into productsizes. A sterile roller is used to roll across the drying fixture.Sufficient pressure must be applied so that the dehydrated tissue is cutalong all of the raised blades or edges of the drying fixture.

After cutting, each tissue graft is placed in a respective “inner”pouch. The inner pouch, which preferably has a clear side and an opaqueside, should be oriented clear side facing up. The tissue graft isplaced in the “inner” pouch so that the texture in the form of text,logo, name, or similar design is facing out through the clear side ofthe inner pouch and is visible outside of the inner pouch. This processis repeated for each separate graft.

Each tissue graft is then given a final inspection to confirm that thereare no tears or holes, that the product size (as cut) is withinapproximately 1 millimeter (plus or minus) of the specified size forthat particular graft, that there are no noticeable blemishes ordiscoloration of the tissue, and that the textured logo or wording isreadable and viewable through the “inner” pouch.

To the extent possible, oxygen is removed from the inner pouch before itis sealed. The inner pouch can be sealed in any suitable manner;however, a heat seal has shown to be effective. In one aspect, afterpackaging, the product is terminally sterilized by radiation, usinggamma or electron beam sterilization with a target dose of 17.5 kGy.Next, each inner pouch is separately packaged in an “outer” pouch forfurther protection, storage, and shipment.

It should be noted that the above process does not require freezing ofthe tissue to kill unwanted cells, to decontaminate the tissue graft, orotherwise to preserve the tissue graft. The dehydrated tissue graftsdescribed herein are designed to be stored and shipped at room orambient temperature without need for refrigeration or freezing.

Product Release (Step 170)

Before the tissue graft is ready for shipment and release to the enduser, all documentation related to manufacture, recovery and donoreligibility are reviewed and deemed acceptable by the quality assurancedepartment and the medical director. Appropriate labeling and chain ofcustody is observed throughout all of the above processes, in accordancewith accepted industry standards and practice. Appropriate clean roomand sterile working conditions are maintained and used, to the extentpossible, throughout the above processes.

II. Applications of Tissue Grafts

These membranes are composed of at least one layer of amnion tissuewhere the epithelium layer has been substantially removed in order toexpose the basement layer to host cells. By removing the epitheliumlayer, cells from the host can more readily interact with thecell-adhesion bio-active molecules located onto top and throughout thebasement membrane. These grafts could be composed of one or severallayers of amnion tissue, combined with chorion or other biocompatiblemembranes to form tissue grafts. The laminin structure of amnion tissueis nearly identical to that of native human oral mucosa tissue. Theyboth include high level of laminin-5, a cell adhesion factor show tobind gingival epithelia-cells, throughout upper portions of the basementmembrane. By creating amnion only, amnion chorion graft, or compositegraft where amnion tissue forms the outer most layer of the membrane,the membranes described herein will be highly suited for use inregenerative procedures involving the oral mucosa. Due to the similarityof mucosa tissue found in oral cavity, nose, throat, vagina, and rectum,it should also be highly effective in regenerative procedures in thoseanatomical regions. Although the laminins found in the basement membranedo not adhere to dermis epithelia cells, and it is unknown if cellsspecific to another anatomical location my adhere to the laminins foundit on the basement membrane of amnion tissue, the amnion based membranescould be effective in broad range of applications including thetreatment of burns, chronic wounds, and as grafts in cardiovascularsurgery.

These membranes can be soaked with a bioactive agent such as a solutioncomposed of naturally occurring growth factors sourced from plateletconcentrates, either using autologous blood collection and separationproducts, or platelet concentrates sourced from expired banked blood;bone marrow aspirate; stem cells derived from concentrated humanplacental cord blood stem cells, concentrated amniotic fluid stem cellsor stem cells grown in a bioreactor; or antibiotics. Here, one or moremembrane layers of the tissue graft absorb the bioactive agent. Uponapplication of the wet tissue graft with bioactive agent to the wound,the bioactive agent is delivered to the wound over time.

The tissue grafts are easy to handle and do not readily break.Additionally, the tissue may composed in such an manner to hold sutures,provide specific rates of resorption based on the requirements of theindication in which it is being used.

The tissue grafts described herein have numerous applications and can beused in a variety of procedures. For example, the tissue grafts can beused in the following perioplastic procedures including the treatment ofrecession defects, increasing gingival tissue height and width, increasethe amount of attached gingival tissue at the gingival margin, andincrease the zone of attached gingival tissue, elimination of a frenumpull, regeneration of lost patella tissue, repair of the Schneiderianmembrane in the sinus cavity, soft tissue around dental implants,vestibuloplasty, and guided tissue regeneration. In oral surgery theycould be used to improve soft tissue outcomes and grow new bone inguided bone regeneration procedures. In applications involving oralmucosa tissue treating mouth sores and oral lesions, and larger replacelarger amounts of mucosa tissue lost through disease or traumaticinjury. These same membranes could also be used in reconstructiveprocedures where the tissue is composed of mucosa including in ear,nose, and throat, urogynecology, and surgical procedures involving therectum and anus.

Methods for using the tissue grafts herein in periodontal applicationsare provided in the Examples.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, and methods described and claimed herein aremade and evaluated, and are intended to be purely exemplary and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. or is at ambienttemperature, and pressure is at or near atmospheric. There are numerousvariations and combinations of reaction conditions, e.g., componentconcentrations, desired solvents, solvent mixtures, temperatures,pressures and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

Evaluation of Tissue Grafts to Repair Gingival Recession Defects

Tissue grafts composed of (5) layers of amniotic membrane with (1) eachlayer having intact epithelium and (2) the epithelial layer removed fromthe first amnion layer to expose the basement layer were evaluated. Theepithelial layer was removed using a specialized instrument (custom cellscraper) according to SOP#220.125, “Removal of the epithelial cell layerfor de-epithelialized products”. This procedure would allow for completeremoval of the epithelial cell layer and exposure of the basementmembrane. After removal of the epithelial cell layer, a representativetissue sample from the processing lot is placed onto a standardmicroscope examination slide. The tissue sample is then stained usingEosin Y Stain. The sample is then covered and allowed to stand. Once anadequate amount of time has passed to allow for staining, visualobservation is done under magnification. The presence of cells andcellular material will appear darker than the areas which have beende-epithelialized.

A total of (10) consenting patients were implanted with the tissuegrafts under initial design specifications. Patient #1 of the case studyevaluation was diagnosed with a Miller's Class I recession defectresulting in surgical decision. The patient displayed a pre-operativerecession defect measuring 3mm at tooth site #5 in the maxillary region.Standard surgical techniques were employed with tissue graft placement(with epithelium) and closure technique. The exposed basement membranewas applied to the gingival, with the fibroblast layer attached to thetooth. Post-operative observations revealed soft tissue closure (flap)recession and soft tissue non-attachment at the (1) week post-operativeinterval.

After post-operative evaluation of patient #1, an assessment was made toaddress the soft tissue non-attachment. The cause of non-attachment wasdue to an interaction of the intact epithelial layer on the tissue graftwith the migration of the patient's oral epithelial layer, which extendsfrom the free gingival margin (FGM) to the gingival sulcus (GS). Theinteraction of the two epithelial tissue surfaces resulted innon-attachment.

Revision surgery was scheduled for patient#1 following the observationalfollow-up visit. A repeat procedure at the same surgical site using thetissue graft without the epithelial layer was scheduled. The epitheliallayer was manually removed intraoperatively using a hand scalpel blade.At the 8 week post-operative interval, patient #1 showed good softtissue attachment with no evidence of soft tissue flap recession.Post-operative recession measurements were taken and documented at 1 mm.All subsequent grafts used in the case study evaluation were manuallyde-epithelialzed intraoperatively. The remaining patients enrolled inthe case study evaluation exhibited no visual evidence of soft tissuenon-attachment and no evidence of soft tissue flap recession.

A second study was conducted to confirm the observations and comparinggrafts prepared using the methods described herein (control materialwith no epithelial layer) and grafts that were prepared without removingthe epithelial layer (test material). All patients were diagnosed withMiller Class I/II recession defects. Success was determined if thepatients obtained 90% root coverage. A total seven patients (15 teeth)were treated with the test material and nine patients (13 teeth) withthe control material. In the control group, 8 of the 13 (62%) teethtreated were deemed successful whereas only 2 of the 15 (13%) teethtreated with the test material had a successful outcome. Although thisstudy was not powered for statistical significance, the date obtainedclearly showed the presence of the epithelial layer resulted in a lowerrate of success.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the compounds, compositions and methods described herein.

Various modifications and variations can be made to the compounds,compositions and methods described herein. Other aspects of thecompounds, compositions and methods described herein will be apparentfrom consideration of the specification and practice of the compounds,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

What is claimed is:
 1. A dehydrated amnion membrane consisting ofnon-fixated, dehydrated modified amnion wherein the modified amnionconsists of a first side which is an exposed basement membrane and asecond side which is a jelly-like fibroblast cellular layer, and whereinthe amnion membrane is dehydrated on or in a heated drying fixture.